9alpha-hydroxy steroids



2,840,580 9u-HYDR0XY STEROIDS David Perlinan, Princeton, James D. Butcher and Josef Fried, New Brunswick, N. L, and Elwood 0. Titus,

Kensington, Md., assignors to 01in Mathieson Chemical Corporation, New York, N. Y., a corporation of Virginia 1 I No Drawing; Application May 24, 1957 Serial No.661,309 I This application isacontinuation-in-part of our parent application Serial No. 283,122, filed April 18, 1952, and

now abandoned.

This invention relates to, and has for its object, the provision of 9a-hydrox'y-Compound S [A -pregnene-9a,17a, 21-triol-3,20-dione] and 21-esters thereof, steroids of the general formula wherein Y is hydrogen, or acyl (particularly the acyl radical of a hydrocarbon carboxylic acid having less than ten carbon atoms).

The steroids of this invention are physiologically active substances which possess glucocorticoid activity. Hence they can be used in lieu of hydrocortisone, for example, in the treatment of rheumatoid arthritis, for which purpose they are administered in the same manner as hydrocortisone with dosage adjusted for the relative activity of the particular steroid.

9a-hydroxy-Compound S is prepared by subjecting Compound S (or a 21-ester thereof) to the action of enzymes of the microorganism Streptomyces atureofaciens under oxidizing conditions. The oxidation can best be eiiected by either including the steroid in the aerobic culture of the microorganism, or by bringing together in an aqueous medium the steroid, air and enzymes of non-proliferating cells of the microorganism.

In general, the conditions of culturing Streptomyces aureofaciens for the purposes of this invention are (except for the inclusion of the steroid to be converted) the same as those of culturing various other Streptomyces for the production of antibiotics and/or vitamin B i. e. the microorganism is aerobically grown in contact with (in or on) a suitable fermentation medium. A suitable medium essentially comprises a source of carbon and energy. The latter may be a carbohydrate (such as sucrose, molasses, glucose, maltose, starch or dextrin), a fatty acid, a fat (such as soybean oil) and/or the'steroid itself. Preferably, however, the medium includes an assimilable source of carbon and energy in addition to the steroid.

The source of nitrogenous factors may be organic (e. g., soybean meal, corn steep liquor, meat extract and/or distillers solubles) or synthetic (i. e., composed of simple, synthesizable organic or inorganic compounds such as ammonium salts, alkali nitrates, amino acids or urea).

An adequate sterile-air supply should be maintained during fermentation, for example by the conventional States Patent fOfice 2,840,580 Patented June 24, 1958 methods of exposing a large surface of the medium to air or by utilizing submerged aerated culture. The steroid may be added to the culture during the incubation period or included in the medium prior to sterilization or inoculation. The preferred range of concentration of the steroid in the culture is about 0.01 to 0.10%., The culture period may vary considerably, the range of about 6 30096 hours being feasible, but not limiting.

The process yields, inter alia, 9a-hydroxy-Compound S, which can, if desired, be esterified by treatment with the desired acid anhydrideor acyl: halide in an organic solvent (preferably an organic base such as pyridine) to yield the corresponding 211-ester;.derivative. The preferred acylating agents are those of hydrocarbon carboxylic acids of less than tencarbon atoms, as exemplified by the acid anhydrides or acyl chlorides of the lower alkanoic acids (e. g., acetic, propionic and enanthic acid), the monocyclic aromatic carboxylic acids (e. g.,benzoic and o, in, orp-toluic acid), the monocyclic aralkanoic acids (e. g., u-toluic and fl -phenylpropionic acid), the lower alkenoic acids, the lower cycloalkanemarboxylic acids and the lower cycloalkene carboxylic acids.

The following examples illustrate the process of this invention: 5

EXAMPLE 1 9u-hyd'roxy-C0rnp0und S (a) FERMENTATION 'An aqueous medium of the following composition is prepared:

Soybean meal t s s gt 15 Glucose g l0 g Soybean oil ml 2.2 Reichsteins Compound S acetate g 0.2 Water liter 7 1 One hundred-milliliter portions of the medium are distributed in fifty SOD-ml. Erlenmeyer flasks, and the flasks are plugged with cotton and sterilized in the usual manner (by autoclaving). When cool, each of the flasks is inoculated with 3 ml. of a vegetative inoculum of Streptomyces aureofaciens NRRL 2209 which has been grown for 48 hours on a soybean meal-glucose medium. The flasks are then placed on a reciprocating shaker four-inch cycles per minute), and agitation as well as aeration is allowed to proceed at 24-25 C. for five days. The contents of the fiasksare pooled and, after the pH of the whole culture is adjusted to 3-4 with H 50 the solids are removed by centrifugation. The aqueous fraction is then extracted three times with an equal volume of chloroform. The chloroform is removed by evaporation under vacuum, leaving a residue of the solid oxidation products (usually containing some lipid material and unoxidized steroid from the culture medium as impurities).

(b) ISOLATION form, then successively with chloroform containing 5,

10, 15 and 20% acetone. The material eluted with (l) chloroform and (2) chloroform containing 5% acetone is found to be essentially Reichsteins S. The material eluted with chloroform containing 10 or more percent acetone, when subjected to paper chromatography, is

The methanol solution re found to contain components having essentially same or slower mobilitythan cortisone, thus indicating microbiological oxidation. The fractions obtained by elution with chloroformcontaining 10% acetone and chloroform containing 15 acetoneareactive in the. liver glycogen tesfofyenningketal. [Endocrinology, 38, 79 1946)]. :sulfuric-acidgives a yellow-green fluorescence characteristic of cortisone and =hydrocortisone. Sin-hydroxy-Compound S is isolated as a crystalline component of the chloroform 15% acetone eluate. It has the 'fOlIOWillgfil'OPel'iitStyMiP. (Block) about 218-220 C.,

after softening at about C.; [a]+l20 (c, 0.15

in ethanol). M

Found: p; 69.19;H-,-s.09. W

ifff EXAMPLE? (a FEBMEN'I'ATION Arabia's-Calculated s g m o c. 69.59; H;- 8.34.

of-i thepfollowing composition is A The procedure of Example l(a).is followed except that the above-described medium isused in place of that given in Example 1.. In addition to the. desired oxidation prod- 4 ucts, about 0.15 pg/ml. of vitamin B is present in the culture filtrate. i a a A H (b) I SOLATION Following the procedure of Example l(b), but using a batch of oxidized products? obtained as described in Example 2(a), substantially the same results are obtained.

Analysis of the crystalline component isolated from the chloroform 15% :acetone eluate gives 9a-hydroxy- Compound S, having the following properties: M; P. (Block) about 218-220 C., after softening at about 204 C.; [a] +l20 (c, 0.15 in ethanol).

Analysis.-Calculated..-for C H O z C, 69.59; H, 8.34. Found: C, 69.51; H, 8.09.

The invention may be otherwise variously embodied within the scope of the appended claims.

What is claimed is:

l. Asteroid of the general formula 

1. A STEROID OF THE GENERAL FORMULA 